Selection of initiation sites for DNA replication in metazoa involves two components: a DNA replication origin and an origin recognition complex (ORC). DNA replication origins are determined by DNA sequences referred to as replicators and by epigenetic parameters such as chromatin structure, nuclear organization, transcription, transcription factors, deoxyribonucleotide pools, DNA topology, and DNA methylation. Consequently, the density of active replication origins can change from an average of one every 10 to 20 kb in the rapidly cleaving embryos of frogs, flies and fish to one every 50 to 300 kb in the differentiated cells of adult organisms. Developmental changes in origin density also occur during specific stages in mammalian development. Thus, metazoan genomes contain many potential initiation sites for DNA replication, but during development, some of these sites are selectively activated while others are suppressed. [unreadable] [unreadable] During the past fiscal year, we have continued to ask, ?How do ORCs recognize DNA replication origins? This process appears to different significantly between yeast and mammals. Yeast ORCs consist of a stable complex of six different subunits that remain bound to chromatin throughout cell division and target specific DNA sequences. In contrast, human ORC consists of a stable core complex [ORC(2-5)] of Orc2 through Orc5 that interacts weakly with Orc1 and Orc6. Nevertheless, the association of Orc1 with ORC(2-5) is essential for prereplication complex assembly and DNA replication. In vitro, however, metazoan ORCs exhibit little affinity for specific DNA sequences other than a preference for asymmetric A:T-rich regions. Nevertheless, in the differentiated cells of mammals and flies, ORCs is localized at specific genomic sites coincident with DNA replication origins. Thus, the ability of ORC to activate a particular replication origin appears to depend on its ability to interact with DNA as it exists within the nucleus, an interaction that appears to be regulated by Orc1. [unreadable] [unreadable] During the past year, we have discovered:[unreadable] [unreadable] 1) the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin. Thus, Orc1 appears to be directly involved in ?origin recognition?, the process of deciding where to initiate DNA replication along the genome. [unreadable] [unreadable] 2) The intracellular level of Orc2 is regulated, and Orc2 contains a single domain that is required in vivo for interaction with any of the other ORC subunits, as well as two nuclear localization signals that are required for accumulation of Orc2 and ORC(2?5) in the nucleus. Moreover, although ORC(2?5) accumulated in the nucleus, only ORC(1?5) was bound to the chromatin fraction, suggesting that Orc1 is required for binding the ORC(2?5) core complex to chromatin.[unreadable] [unreadable] 3) Orc1 undergoes cell cycle dependent changes (ubiquitination, phosphorylation, and in some cases degradation) that activate ORC during the transition from M to G1-phase, and suppress it during the transition from S to M-phase. Furthermore, unmodified Orc1 can induce apoptosis. Thus, the same modifications that prevent Orc1 from participating in DNA replication also neutralized its ability to induce apoptosis, suggesting that failure to regulate ORC activity during mammalian development could result in cell death.[unreadable] [unreadable]